Histoplasma capsulatum is a dimorphic fungus which can cause a respiratory or disseminated disease in humans. The work proposed here is directed at identifying characteristics which enable this organism to be a " successful" pathogen. Requirements for intracellular survival will be evaluated by a systematic examination of a variety of strains, including matched virulent/avirulent strain pairs. In contrast to other laboratories which are investigating how macrophages respond when confronted with H. capsulatum, this proposal focuses on how H. capsulatum has adapted to survive in the potentially hostile environment within macrophages. The first issue addressed is whether virulent H. capsulatum yeasts are better able to survive within macrophages than avirulent yeasts. Alternatively, avirulent yeasts may survive but fail to multiply or perhaps remain metabolically inactive. These possibilities will be examined through a variety of assays using radiolabel incorporation, vital staining, or colony formation as indicators. Later studies explore the mechanisms by which virulent yeasts survive and proliferate in macrophages. In P388D1 cells, phagosomes containing this organism appear to fuse with lysosomes. This will be confirmed by electron microscopy and evaluated in resident peritoneal and alveolar macrophages as well. Since phagosome-lysosome fusion occurs, H. capsulatum must either resist or inactivate the fungicidal mechanisms found in lysosomes. The sensitivity of this organism to oxidative killing mechanisms will be assessed by exposure to exogeneously generated products of the oxidative burst. Likewise, vulnerability to non-oxidative killing mechanisms will be studied in rat macrophages, which are exceptionally adept in these activities. Effects of the fungicidal lysosomal peptides MCP-1 and MCP-2 on this yeast will also be examined. One means of inactivating intracellular fungicidal mechanisms would be to alter the phagolysosomal environment. While phagosomes normally acidify, preliminary experiments using ph- sensitive fluorescein isothiocyanate indicate that those containing virulent yeasts do not. These studies will be confirmed using other ph-sensitive strains as well as probes which become fluorescent only after cleavage by ph-sensitive lysosomal enzymes. Recent reports have suggested that phagolysosomes contain very little calcium; therefore, fura-2 will be used to determine the concentration of calcium in H. capsulatum phagolysosomes. Additional experiments will address the possibility that this yeast has adapted to environments low in calcium by releasing calcium-binding proteins.